The present invention relates generally to materials and methods for use in immunological procedures for isolation and quantitative detection of a sweet tasting polypeptide known as thaumatin, from biological fluids. More specifically, the invention relates to monoclonal antithaumatin antibodies, produced by novel hybridoma cell lines (as exemplified by A.T.C.C. HB-8921 and A.T.C.C. HB-8922) and to uses of these antibodies in isolation of thaumatin through affinity purification techniques, in assays for detection of thaumatin, and in immunological techniques for study of sweet thaumatin-like polypeptides.
Thaumatin is an extremely sweet-tasting protein produced in the arils of the fruit of the African shrub Thaumatococcus daniellii Benth. The fruit traditionally has been used in West Africa as a sweetener of palm wine, corn, bread and sour fruit. Thaumatin, which is about 5000 times sweeter than sucrose on a weight basis, is produced in at least five forms: thaumatins I, II, a, b and c. These proteins, named in their order of elution from an ion exchange column [Higgenbotham, et al., in Sensory Properties of Foods (Birch, et al., eds.), London: Applied Sciences, pp. 129-149 (1977)], have molecular weights of approximately 22 kilodaltons.
Thaumatin I and II are non-toxic proteins, are low calorie and non-cariogenic, and elicit profound taste responses suggesting a stable interaction between these proteins and human taste buds. Therefore, thaumatin has potential for use as a sugar substitute, food additive, a sweetness receptor probe and a tool for further elucidation of the taste response.
A plentiful supply of pure thaumatin is required to utilize the protein as a possible food additive and research tool. Because T. daniellii requires a tropical climate and insect pollination for successful fruit propagation, there are considerable difficulties involved in greenhouse cultivation of the fruit. For these reasons, considerable effort has been directed toward the introduction of genes into recombinant microorganisms enabling them to synthesize thaumatin. One research group reported an amino acid sequence for thaumatin I. Iyengar, et al., Eur.J.Biochem., 96, 193-204 (1979)]. The research group also reported the successful cloning of a gene for thaumatin II from messenger RNA-dervived cDNA [Edens, et al., Gene, 18, 1-12 (1982)]. The Edens, et al. reference cited above notes that a polypeptide having the native sequence of preprothaumatin II has been microbially produced. More specifically, the reference and European Patent Application Nos. 54,330 and 54,331 disclose cDNA sequences coding for native mature thaumtin II and preprothaumatin II and also disclose cloning vehicles comprising the DNA sequences for use in transformation in microorganisms.
In co-owned and copending U.S. patent application No. 540,634 filed Oct. 11, 1983, the successful synthesis of "manufactured" genes coding for thaumatin I having a primary structural conformation duplicating the sequence provided in Iyengar, et al. was disclosed along with their expression in bacterial and yeast hosts.
Of interest to the background of the invention is current research focused on hybridoma techniques for producing tumor cell lines which will manufacture highly specific monoclonal antibodies to a selected antigenic substance. Techniques for the production of monoclonal antibodies are generally well known in the art. Typical descriptions of these procedures may be found in Wands, J. R., and Zurawski, V. R., Gastroenterology 80:225 (1981); Marshak-Rothstein, etal., J. Immunol. 122:2491 (1979); and Oi, V. T. and L. A. Herzenberg, "Immunoglobulin Producing Hybrid," Mishell, B. B. and S. M. Shiigi (eds.) Selected Methods in Cellular Immunology, San Francisco: W. H. Freeman Publishing, 1979. Briefly summarized, lymphocytes removed from the spleen of an animal previously injected with the antigen of interest are induced to fuse with myeloma cells in the presence of polyethylene glycol. Thousands of "hybrid" myeloma cells are produced from the fusion. The supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture, it is cloned by limiting dilutions, and the clones are individually assayed for supernatant activity.
Due to the highly specific nature of their immunological properties, monoclonal antibodies developed according to hybridoma techniques have been proposed for use as diagnostic reagents, therapeutic agents, and agents for affinity purification of specifically cross-reactive antigenic proteins from crude sources. See, e.g., Trends in Biotechnology, Vol. 3, No. 7 (July, 1985) and U.S. Pat. Nos. 4,465,624, 4,514,505 and 4,514,507.
While there exists a substantial need for specific monoclonal antibodies for use in detecting, isolating, purifying and studying thaumatin and thaumatin-like polypeptide molecules, there have been no reports of the successful use of hybridoma techniques in obtaining monoclonal antibodies to thaumatin.